Project Summary Mitochondrial DNA (mtDNA) mutations accumulate with age and are present at pathological levels in affected tissues of certain mitochondrial disorders and degenerative diseases (reviewed in [1, 2]). mtDNA mutations are susceptible to mitochondrial quality control, the process of selective autophagy of defective mitochondria, as evidenced by observations that stimulation of mitochondrial autophagy can reduce levels of these mutations in model organisms [3-6] and cell culture [7, 8] and that defects in mitochondrial quality control correlate with increased levels of mtDNA mutations [1, 2, 9]. However, the susceptibility of mtDNA to quality control is poorly understood because it is influenced by multiple complex processes, including the varied sensitivity of quality control to different types of defects in mtDNA gene products, the exchange of gene products between organelles through fission and fusion, the impact of mutations on mtDNA replication, and stochasticity of mtDNA turn-over and inheritance. The proposed work seeks to understand how the nature of different mtDNA mutations determines whether they tend to increase or decrease in frequency within the cell (this frequency is known as the level of heteroplasmy). To do this, I have developed a simple cell culture system that enables the experimenter to tune the mtDNA mutation rate and copy number per cell, as shown in preliminary data. Using this system, I am generating a library of cells, each containing one or few new mtDNA mutations present at intermediate frequency within the cell. Changes in intracellular frequencies of mutations in response to different treatments, particularly autophagy stimulation, can then be tracked using DNA sequencing. Experiments to elucidate mechanisms mediating these changes take advantage of greatly improved methods of mitochondrial purification recently developed in the Sabatini Lab. The specific aims are: I) To elucidate the prevalence and mechanisms of mtDNA mutations that bias mtDNA replication. II) To understand how mitochondrial quality control acts on different types of mtDNA mutations. The key novelty of the approach is to measure changes in frequency of many different kinds of mtDNA mutations in high throughput. Prior studies focused on a small set of mutations corresponding to drug resistance markers or relatively-common mitochondrial diseases [12, 13]. In contrast, the goal here is to obtain similar data for thousands of mutations in a single cell type, approaching saturation-level mutagenesis of the ~16.5kb mammalian mitochondrial genome. The proposed work aims to elucidate the factors limiting efficiency of mitochondrial quality control with respect to diverse forms of mtDNA-encoded mitochondrial dysfunction. Understanding these limitations may suggest what renders cells more or less prone to the decline of mitochondrial function in aging, degenerative disease and mtDNA disorders.